The present invention relates to subtilisin protease conjugates and compositions comprising the conjugates which have decreased immunogenicity relative to their corresponding parent proteases.
Enzymes make up the largest class of naturally occurring proteins. One class of enzyme includes proteases which catalyze the hydrolysis of other proteins. This ability to hydrolyze proteins has typically been exploited by incorporating naturally occurring and protein engineered proteases into cleaning compositions, particularly those relevant to laundry applications.
In the cleaning arts, the mostly widely utilized of these proteases are the serine proteases. Most of these serine proteases are produced by bacterial organisms while some are produced by other organisms, such as fungi. See Siezen et al., xe2x80x9cHomology Modelling and Protein Engineering Strategy of Subtilases, the Family of Subtilisin-Like Serine Proteasesxe2x80x9d, Protein Engineering, Vol. 4, No. 7, pp. 719-737 (1991). Unfortunately, the efficacy of the wild-type proteases in their natural environment frequently does not translate into the unnatural cleaning composition environment. Specifically, protease characteristics such as, for example, thermal stability, pH stability, oxidative stability, and substrate specificity are not necessarily optimized for utilization outside the natural environment of the protease.
Several approaches have been employed to alter the wild-type amino acid sequence of serine proteases with the goal of increasing the efficacy of the protease in the unnatural wash environment. These approaches include the genetic redesign and/or chemical modification of proteases to enhance thermal stability and to improve oxidation stability under quite diverse conditions.
However, because such proteases are foreign to mammals, they are potential antigens. As antigens, these proteases cause immunogenic and/or allergenic responses (herein collectively described as immunogenic responses) in mammals. In fact, sensitization to serine proteases has been observed in environments wherein humans are regularly exposed to the proteases. Such environments include manufacturing facilities, wherein workers are exposed to the proteases through such vehicles as uncontrolled dust or aerosolization. Aerosolization can result by the introduction of the protease into the lung, which is the route of protease exposure which causes the most dangerous response. Protease sensitization can also occur in the marketplace, wherein consumers"" repeated use of products containing proteases may cause an immunogenic response.
Furthermore, while genetic redesign and chemical modification of proteases has been prominent in the continuing search for more highly effective proteases for laundry applications, such proteases have been minimally utilized in personal care compositions and light duty detergents. A primary reason for the absence of these proteases in products such as, for example, soaps, gels, body washes, and shampoos, is due to the aforementioned problem of human sensitization leading to undesirable immunogenic responses. It would therefore be highly advantageous to provide a personal care composition which provides the cleansing properties of proteases without the provocation of an immunogenic response.
Presently, immunogenic responses to proteases may be minimized by immobilizing, granulating, coating, or dissolving chemically modified proteases to avoid their becoming airborne. These methods, while addressing consumer exposure to airborne proteases, still present the risks associated with extended tissue contact with the finished composition and worker exposure to protease-containing dust or aerosol during manufacturing.
In the medical field, suggestions have been made to diminish the immunogenicity of enzymes through yet another method. This method involves attaching polymers to enzymes. See. e.g., U.S. Pat. No. 4,179,337, Davis, et al., issued Dec. 18, 1979 and PCT Application WO 96/17929, Olsen, et al., published Jun. 13, 1996.
One approach toward decreasing the immunogenic activity of a protease is through alleviation of the immunogenic properties of epitopes. Epitopes are those amino acid regions of an antigen which evoke an immune response through the binding of antibodies or the presentation of processed antigens to T cells via a major histocompatibility complex protein (MHC). Changes in the epitopes can affect their efficiency as an antigen. See Walsh, B. J. and M. E. H. Howden, xe2x80x9cA Method for the Detection of IgE Binding Sequences of Allergens Based on a Modification of Epitope Mappingxe2x80x9d, Journal of Immunological Methods, Vol. 121, pp. 275-280 (1989).
The present inventors have discovered that those serine proteases commonly known as subtilisins, including subtilisin BPNxe2x80x2, have prominent epitope regions at amino acid positions 70-84, 103-126, and 217-252 corresponding to subtilisin BPNxe2x80x2. The present inventors have herein chemically modified such subtilisins at one or more of these epitope regions to alleviate the immunogenic properties of the protease. In so doing, the active site of the protease is minimally affected. The present inventors have therefore discovered subtilisin-like proteases which evoke a decreased immunogenic response yet maintain their activity as an efficient and active protease. Accordingly, the present protease conjugates are suitable for use in several types of compositions including, but not limited to, laundry, dish, hard surface, skin care, hair care, beauty care, oral care, and contact lens compositions.
The present invention relates to subtilisin protease conjugates comprising a protease moiety and one or more addition moieties wherein:
(a) the protease moiety has a modified amino acid sequence of a parent amino acid sequence, the parent amino acid sequence comprising a first epitope region, a second epitope region, and a third epitope region, wherein the modified amino acid sequence comprises a substitution by a substituting amino acid at one or more positions in one or more of the epitope regions wherein:
(i) when a substitution occurs in the first epitope region, the substitution occurs at one or more positions corresponding to positions 70-84 of subtilisin BPNxe2x80x2;
(ii) when a substitution occurs in the second epitope region, the substitution occurs at one or more positions corresponding to positions 103-126 of subtilisin BPNxe2x80x2; and
(iii) when a substitution occurs in the third epitope region, the substitution occurs at one or more positions corresponding to positions 217-252 of subtilisin BPNxe2x80x2; and
(b) wherein each of the addition moieties is covalently attached to one of the substituting amino acids present on the protease moiety and has the structure: 
wherein X is selected from the group consisting of nil and a linking moiety; R1 is selected from the group consisting of nil, a first polypeptide, and a first polymer; and R2 is selected from the group consisting of nil, a second polypeptide, and a second polymer; wherein at least one of X, R1, and R2 is not nil. The present invention further relates to cleaning and personal care compositions comprising such protease conjugates.
The essential components of the present invention are herein described below. Also included are non-limiting descriptions of various optional and preferred components useful in embodiments of the present invention.
The present invention can comprise, consist of, or consist essentially of any of the required or optional components and/or limitations described herein.
All percentages and ratios are calculated by weight unless otherwise indicated. All percentages are calculated based on the total composition unless otherwise indicated.
All component or composition levels are in reference to the active level of that component or composition, and are exclusive of impurities, for example, residual solvents or by-products, which may be present in commercially available sources.
All documents referred to herein, including all patents, patent applications, and printed publications, are hereby incorporated by reference in their entirety.
Referred to herein are trade names for materials including, but not limited to, enzymes. The inventors herein do not intend to be limited by materials under a certain trade name. Equivalent materials (e.g., those obtained from a different source under a different name or catalog (reference) number) to those referenced by trade name may be substituted and utilized in the protease conjugates and compositions herein.
As used herein, abbreviations will be used to describe amino acids. Table I provides a list of abbreviations used herein:
As used herein, the term xe2x80x9cmutationxe2x80x9d refers to alterations in gene sequences and amino acid sequences produced by those gene sequences. Mutations include deletions, substitutions, and additions of amino acid residues to the wild-type protein sequence.
As used herein, the term xe2x80x9cparentxe2x80x9d refers to an enzyme, wild-type or variant.
As used herein, the term xe2x80x9cwild-typexe2x80x9d refers to a protein, for example a protease or other enzyme, produced by unmutated organisms.
As used herein, the term xe2x80x9cvariantxe2x80x9d means an enzyme having an amino acid sequence which differs from that of the corresponding wild-type enzyme due to the genetic mutation of the nucleotide sequences coding for that enzyme or the mutation of the wild-type enzyme itself.
As used herein, all polymer molecular weights are expressed as weight average molecular weights.
As referred to herein, while the conjugates of the present invention are not limited to those comprising subtilisin BPNxe2x80x2 and variants thereof, all amino acid numbering is with reference to the amino acid sequence for subtilisin BPNxe2x80x2 which is represented by SEQ ID NO:1. The amino acid sequence for subtilisin BPNxe2x80x2 is further described by Wells et al., Nucleic Acids Research, Vol. II, pp. 7911-7925 (1983).
The protease conjugates of the present invention are compounds which comprise a protease moiety and one or more addition moieties, wherein the protease moiety and the addition moieties are connected via covalent bonding.
Protease Moieties
The protease moieties herein have a modified amino acid sequence of a parent amino acid sequence. The parent amino acid sequences herein are serine proteases, either wild-type or variants thereof. As used herein, the term xe2x80x9cserine proteasexe2x80x9d means a protease which has at least 50%, and preferably 80%, amino acid sequence identity with the sequences of one or more subtilisin-like serine proteases. Wild-type subtilisin-like serine proteases are produced by, for example, Bacillus alcalophilus, Bacillus amyloliquefaciens, Bacillus amylosaccharicus, Bacillus licheniformis, Bacillus lentus, and Bacillus subtilis microorganisms. A discussion relating to subtilisin-like serine proteases and their homologies may be found in Siezen et al., xe2x80x9cHomology Modelling and Protein Engineering Strategy of Subtilases, the Family of Subtilisin-Like Serine Proteasesxe2x80x9d, Protein Engineering, Vol. 4, No. 7, pp. 719-737 (1991).
Preferred parent amino acid sequences for use herein include, for example, those obtained from Bacillus amyloliquefaciens, Bacillus licheniformis, and Bacillus subtilis, subtilisin BPN, subtilisin BPNxe2x80x2, subtilisin Carlsberg, subtilisin DY, subtilisin 309, proteinase K, and thermitase, including A/S Alcalase(copyright) (commercially available from Novo Industries, Copenhagen, Denmark), Esperase(copyright) (Novo Industries), Savinase(copyright) (Novo Industries), Maxatase(copyright) (commercially available from Gist-Brocades, Delft, Netherlands), Maxacal(copyright) (Gist-Brocades), Maxapem 15(copyright) (Gist-Brocades), and variants of the foregoing. Especially preferred proteases for use herein include those obtained from Bacillus amyloliquefaciens and variants thereof. The most preferred proteases for use as protease moieties herein are subtilisin BPNxe2x80x2 and variants thereof.
Especially preferred variants of subtilisin BPNxe2x80x2, hereinafter referred to as xe2x80x9cProtease Axe2x80x9d, for use as parent amino acid sequences herein are disclosed in U.S. Pat. No. 5,030,378, Venegas, issued Jul. 9, 1991, as characterized by the subtilisin BPNxe2x80x2 amino acid sequence with the following mutations:
(a) Gly at position 166 is substituted with an amino acid residue selected from Asn, Ser, Lys, Arg, His, Gln, Ala and Glu; Gly at position 169 is substituted with Ser; and Met at position 222 is substituted with an amino acid residue selected from Gln, Phe, His, Asn, Glu, Ala and Thr; or
(b) Gly at position 160 is substituted with Ala, and Met at position 222 is substituted with Ala.
Additionally preferred variants of subtilisin BPNxe2x80x2, hereinafter referred to as xe2x80x9cProtease Bxe2x80x9d, for use as parent amino acid sequences herein are disclosed in EP-B-251,446, assigned to Genencor International, Inc., published Jan. 7, 1988, granted Dec. 28, 1994, as characterized by the wild-type subtilisin BPNxe2x80x2 amino acid sequence with mutations at one or more of the following positions: Tyr21, Thr22, Ser24, Asp36, Ala45, Ala48, Ser49, Met50, His67, Ser87, Lys94, Val95, Gly97, Ser101, Gly102, Gly103, Ile107, Gly110, Met124, Gly127, Gly128, Pro129, Leu135, Lys170, Tyr171, Pro172, Asp197, Met199, Ser204, Lys213, Tyr214, Gly215, and Ser221; or two or more of the positions listed above combined with one or more mutations at positions selected from Asp32, Ser33, Tyr104, Ala152, Asn155, Glu156, Gly166, Gly169, Phe189, Tyr217, and Met222.
Other preferred subtilisin BPNxe2x80x2 variants for use as parent amino acid sequences herein are hereinafter referred to as xe2x80x9cProtease Cxe2x80x9d, and are described in WO 95/10615, assigned to Genencor International Inc., published Apr. 20, 1995, as characterized by the wild-type subtilisin BPNxe2x80x2 amino acid sequence with a mutation to position Asn76, in combination with mutations in one or more other positions selected from Asp99, Ser101, Gln103, Tyr104, Ser105, Ile107, Asn109, Asn123, Leu126, Gly127, Gly128, Leu135, Glu156, Gly166, Glu195, Asp197, Ser204, Gln206, Pro210, Ala216, Tyr217, Asn218, Met222, Ser260, Lys265, and Ala274.
Other preferred subtilisin BPNxe2x80x2 variants for use as parent amino acid sequences herein, hereinafter referred to as xe2x80x9cProtease Dxe2x80x9d, are described in U.S. Pat. No. 4,760,025, Estell et al., Jul. 26, 1988, as characterized by the wild-type subtilisin BPN"" amino acid sequence with mutations to one or more amino acid positions selected from the group consisting of Asp32, Ser33, His64, Tar104, Asn155, Glu156, Gly166, Gly169, Phe189, Tar217, and Met222.
The more preferred proteases for use as parent amino acid sequences herein are selected from the group consisting of Alcalase(copyright), subtilisin BPNxe2x80x2, Protease A, Protease B, Protease C, and Protease D, with Protease D being the most preferred.
In accordance with the present invention, the parent amino acid sequence is substituted at one or more of the parent amino acid residues with a substituting amino acid to produce a (precursor to a) protease moiety suitable for attachment with one or more of the present addition moieties. The substitution should be made at one or more positions in one or more of the epitope regions which have been discovered by the present inventors. The present inventors have discovered three epitope regions, one occurring at positions 70-84 corresponding to subtilisin BPNxe2x80x2 (the first epitope region), one occurring at positions 103-126 corresponding to subtilisin BPNxe2x80x2 (the second epitope region), and one occurring at positions 217-252 of subtilisin BPNxe2x80x2 (the third epitope region). In another embodiment of the invention, the protease moiety comprises a substitution at one or more positions in two or more of the epitope regions (i.e., one or more substitutions occurring in each of two or all three of the epitope regions). In yet another embodiment of the invention, the protease comprises a substitution at one or more positions in each of the three epitope regions (i.e., one or more substitutions occurring in each of all three of the epitope regions). Most preferably, the parent amino acid sequence is substituted at one or more of the parent amino acid residues wherein at least one of the substitutions occurs in the first epitope region.
Wherein a substitution occurs in the first epitope region, the substitution occurs at one or more of positions 70-84, more preferably positions one or more of positions 73-81, and most preferably at position 78. Wherein a substitution occurs in the second epitope region, the substitution occurs at one or more of positions 106-126, more preferably one or more of positions 106-120, and most preferably at position 116. Wherein a substitution occurs in the third epitope region, the substitution occurs at one or more of positions 217-254, more preferably one or more of positions 236-254, and most preferably at position 240.
In order to best achieve selective attachment (i.e., selective attachment in one or more of the epitope regions) of one or more addition moieties of the present invention to the protease moiety, the substitution should be with a substituting amino acid which does not occur in (is unique to) the parent amino acid sequence. In this respect, any substituting amino acid which is unique to the parent amino acid sequence may be utilized. For example, because a cysteine residue does not occur in the wild-type amino acid sequence for subtilisin BPNxe2x80x2, a substitution of subtilisin BPNxe2x80x2 with one or more cysteine residues in one or more of the epitope regions is suitable for the present invention. Wherein a cysteine residue occurs outside the epitope regions of the parent amino acid sequence, it is preferable to substitute another amino acid residue for in each of those positions to enable selective coupling with one or more addition moieties in the epitope region(s). Cysteine is the most preferred substituting amino acid for substitution in one or more of the epitope regions.
Other preferred substituting amino acids include lysine. Wherein the substituting amino acid is lysine, it is preferred to mutate lysine residues occurring outside the epitope regions of the parent amino acid sequence to another amino acid residue such that functionalization of one or more of the lysine residues in the epitope regions is selective. For example, a lysine residue occurs at position 237 of subtilisin BPN"" which is in the third epitope region. Site-selective mutation of all other lysine residues occurring in the subtilisin BPN"" sequence may be performed followed by selective functionalization of the lysine residue in the third epitope region with an addition moiety. Alternatively, positions in the epitope regions may be mutated to lysine followed by selective functionalization at those positions by a polymer moiety.
Addition Moieties
The protease conjugates of the present invention further comprise one or more addition moieties wherein each of the addition moieties is covalently attached to one of the substituting amino acids present in one of the epitope regions and has the structure: 
wherein X is selected from nil and a linking moiety; R1 is selected from the group consisting of nil, a first polypeptide, and a first polymer; and R2 is selected from the group consisting of nil, a second polypeptide, and a second polymer, wherein at least one of X, R1, and R2 is not nil.
Preferably, from 1 to about 15, more preferably from about 2 to about 10, and most preferably from about 1 to about 5 addition moieties comprise the protease conjugate.
Wherein R1 and R2 are each, independently, polypeptide moieties or polymer moieties, R1 and R2 may be equivalent or different. Preferably, wherein R1 is a polypeptide moiety, R2 is selected from nil and a polypeptide moiety, and is most preferably nil. Most preferably, wherein R1 is a polypeptide moiety, R2 is selected from nil and an equivalent polypeptide moiety, and is most preferably nil. Preferably, wherein R1 is a polymer moiety, R2 is selected from nil and a polymer moiety. Most preferably, wherein R1 is a polymer moiety, R2 is selected from nil and an equivalent polymer moiety. Wherein at least one of R1 and R2 are respectively, the first polymer and the second polymer, then X is preferably not nil.
Polypeptide Moieties
The polypeptide moieties described herein include those comprising two or more amino acid residues. Preferred polypeptide moieties are selected from proteins, including enzymes. Preferred enzymes include proteases, cellulases, lipases, amylases, peroxidases, microperoxidases, hemicellulases, xylanases, phospholipases, esterases, cutinases, pectinases, keratinases, reductases (including, for example, NADH reductase), oxidases, phenoloxidases, lipoxygenases, ligninases, pullulanases, tannases, pentosanases, malanases, xcex2-glucanases, arabinosidases, hyaluronidase, chondroitinase, laccases, transferases, isomerases (including, for example, glucose isomerase and xylose isomerase), lyases, ligases, synthetases, and fruit-based enzymes (including, for example, papain). More preferred enzymes for use as polypeptide moieties include proteases, cellulases, amylases, lipases, and fruit-based enzymes, with proteases being even more preferred.
Examples of lipases for use as a polypeptide moiety include those derived from the following microorganisms: Humicola, Pseudonomas, Fusarium, Mucor, Chromobacterium, Aspergillus, Candida, Geotricum, Penicillium, Rhizopus, and Bacillus.
Examples of commercial lipases include Lipolase(copyright), Lipolase Ultra(copyright), Lipozyme(copyright), Palatase(copyright), Novozym435(copyright), and Lecitase(copyright) (all of which are commercially available from Novo Nordisk A/S, Copenhagen, Denmark), Lumafast(copyright) (commercially available from Genencor, Int., Rochester, N.Y.), and Lipomax(copyright) (Genencor, Int.).
Examples of proteases for use as the polypeptide moiety include serine proteases, chymotrypsin, and elastase-type enzymes. The most preferred proteases for use as a polypeptide moiety include serine proteases, as were defined herein above in the discussion of xe2x80x9cprotease moietiesxe2x80x9d.
Most preferably, wherein the polypeptide moiety is a serine protease, the polypeptide moiety carries, independently, the definition of a protease moiety as described herein above, i.e., the polypeptide moiety has a modified amino acid sequence of a parent amino acid sequence in one or more of the epitope regions as described herein above (which parent amino acid sequence may be referred to as a xe2x80x9csecondxe2x80x9d parent amino acid sequence). In this instance, one of the linking moiety (wherein the linking moiety is not nil) or the protease moiety (wherein the linking moiety is nil) is covalently attached to the polypeptide moiety through one of the substituting amino acids present in one of the epitope regions of the polypeptide moiety. Wherein the polypeptide moiety is a serine protease, the same preferred, more preferred, and most preferred groupings apply as are described herein above for protease moieties and their corresponding parent amino acid sequences.
Most preferably, wherein the polypeptide moiety is a serine protease, the polypeptide moiety and the protease moiety are equivalent moieties. In this instance, the polypeptide moiety and the protease moiety are preferably attached through a disulfide bridge, wherein X is nil, and most preferably, R2 is nil.
Polymer Moieties
The addition moieties herein may comprise a polymer moiety. Examples of suitable polymer moieties include polyalkylene oxides, polyalcohols, polyvinyl alcohols, polycarboxylates, polyvinylpyrrolidones, polyamino acids, celluloses, dextrans, starches, glycogen, agaroses, guar gum, pullulan, inulin, xanthan gum, carrageenan, pectin, biopolymers, alginic acid hydrosylates, and hydrosylates of chitosan. Preferred polyalkylene oxides include polyethylene glycols, methoxypolyethylene glycols, and polypropylene glycols. Preferred dextrans include carboxymethyldextrans. Preferred celluloses include methylcellulose, carboxymethylcellulose, ethylcellulose, hydroxyethyl cellulose, carboxyethyl cellulose, and hydroxypropylcellulose. Preferred starches include hydroxyethyl starches and hydroxypropyl starches. The more preferred polymers are polyalkylene oxides. The most preferred polymer moiety is polyethylene glycol.
Wherein R1 and R2 are each, independently, polymer moieties, R1 and R2 preferably has a collective molecular weight (i.e., molecular weight of R1 plus molecular weight of R2) of from about 0.5 kD (kilodaltons) to about 40 kD, more preferably from about 0.5 kD to about 20 kD, and most preferably from about 1 kD to about 10 kD.
Wherein R1 and R2 are each polymer moieties, R1 and R2 each, independently, preferably have a molecular weight of 0.25 kD to about 20 kD, more preferably from about 0.5 kD to about 10 kD, and most preferably from about 0.5 kD to about 5 kD.
Wherein R1 and R2 are each polymer moieties, the ratio of the molecular weights of R1 and R2 preferably ranges from about 1:10 to about 10:1, more preferably from about 1:5 to about 5:1, and most preferably from about 1:3 to about 3:1.
Wherein R1 is a polymer moiety and R2 is nil, R1 preferably has a molecular weight of from about 0.5 kD to about 40 kD, more preferably from about 0.5 kD to about 20 kD, and most preferably from about 1 kD to about 10 kD.
Linking Moieties
As used herein, X may be (nil or) a linking moiety which is covalently attached to a polypeptide moiety or a polymer moiety and is also covalently attached to a single substituting amino acid present in one of the epitope regions of the protease moiety. The linking moiety is any small molecule, i.e., a molecule having a molecular weight of less than about 800, preferably less than about 400, and more preferably less than about 300. The most preferred linking moieties include those capable of being covalently bound to a cysteine residue or a lysine residue, most preferably a cysteine residue. Examples of linking moieties and related chemistry are disclosed in U.S. Pat. No. 5,446,090, Harris, issued Aug. 29, 1995; U.S. Pat. No. 5,171,264, Merrill, issued Dec. 15, 1992; U.S. Pat. No. 5,162,430, Rhee et al., issued Nov. 10, 1992; U.S. Pat. No. 5,153,265, Shadle et al., issued Oct. 6, 1992; U.S. Pat. No. 5,122,614, Zalipsky, issued Jun. 16, 1992; Goodson et al., xe2x80x9cSite-Directed Pegylation of Recombinant Interleukin-2 at its Glycosylation Sitexe2x80x9d, Biotechnology, Vol. 8, No. 4, pp. 343-346 (1990); Kogan, xe2x80x9cThe Synthesis of Substituted Methoxy-Poly(ethylene glycol) Derivatives Suitable for Selective Protein Modificationxe2x80x9d, Synthetic Communications, Vol. 22, pp. 2417-2424 (1992); and Ishii et al., xe2x80x9cEffects of the State of the Succinimido-Ring on the Fluorescence and Structural Properties of Pyrene Maleimide-Labeled xcex1xcex1-Tropomyosinxe2x80x9d, Biophysical Journal, Vol. 50, pp. 75-80 (1986). The most preferred linking moiety is substituted (for example, alkyl) or unsubstituted succinimide.
The protease moieties are prepared by mutating the nucleotide sequences that code for a parent amino acid sequence. Such methods are well-known in the art; a non-limiting example of one such method is set forth below:
A phagemid (pSS-5) containing the wild-type subtilisin BPNxe2x80x2 gene (Mitchison, C. and J. A. Wells, xe2x80x9cProtein Engineering of Disulfide Bonds in Subtilisin BPNxe2x80x2xe2x80x9d, Biochemistry, Vol. 28, pp. 4807-4815 (1989) is transformed into Escherichia coli dut- ung- strain CJ236 and a single stranded uracil-containing DNA template is produced using the VCSM13 helper phage (Kunkel et al., xe2x80x9cRapid and Efficient Site-Specific Mutagenesis Without Phenotypic Selectionxe2x80x9d, Methods in Enzymology, Vol 154, pp. 367-382 (1987), as modified by Yuckenberg et al., xe2x80x9cSite-Directed in vitro Mutagenesis Using Uracil-Containing DNA and Phagemid Vectorsxe2x80x9d, Directed Mutagenesisxe2x80x94A Practical Approach, McPherson, M. J. ed., pp. 27-48 (1991). Primer site-directed mutagenesis modified from the method disclosed in Zoller, M. J., and M. Smith, xe2x80x9cOligonucleotidexe2x80x94Directed Mutagenesis Using M13xe2x80x94Derived Vectors: An Efficient and General Procedure for the Production of Point Mutations in any Fragment of DNAxe2x80x9d, Nucleic Acids Research, Vol. 10, pp. 6487-6500 (1982) is used to produce all mutants (essentially as presented by Yuckenberg et al., supra).
Oligonucleotides are made using a 380B DNA synthesizer (Applied Biosystems Inc.). Mutagenesis reaction products are transformed into Escherichia coli strain MM294 (American Type Culture Collection E. coli 33625). All mutations are confirmed by DNA sequencing and the isolated DNA is transformed into the Bacillus subtilis expression strain PG632 (Saunders et al., xe2x80x9cOptimization of the Signal-Sequence Cleavage Site for Secretion from Bacillus subtilis of a 34-Amino Acid Fragment of Human Parathyroid Hormonexe2x80x9d, Gene, Vol. 102, pp. 277-282 (1991) and Yang et al., xe2x80x9cCloning of the Neutral Protease Gene of Bacillus subtilis and the Use of the Cloned Gene to Create an in vitroxe2x80x94Derived Deletion Mutationxe2x80x9d, Journal of Bacteriology, Vol. 160, pp. 15-21 (1984).
Fermentation is as follows. Bacillus subtilis cells (PG632) containing the protease of interest are grown to mid-log phase in one liter of LB broth containing 10 g/L glucose, and inoculated into a Biostat C fermentor (Braun Biotech, Inc., Allentown, Pa.) in a total volume of 9 liters. The fermentation medium contains yeast extract, casein hydrosylate, solublexe2x80x94partially hydrolyzed starch (Maltrin M-250), antifoam, buffers, and trace minerals (see xe2x80x9cBiology of Bacilli: Applications to Industryxe2x80x9d, Doi, R. H. and M. McGloughlin, eds. (1992)). The broth is kept at a constant pH of 7.5 during the fermentation run. Kanamycin (50 xcexcg/mL) is added for antibiotic selection of the mutagenized plasmid. The cells are grown for 18 hours at 37xc2x0 C. to an A600 of about 60 and the product harvested.
The fermentation broth is taken through the following steps to obtain pure protease. The broth is cleared of Bacillus subtilis cells by tangential flow against a 0.16 xcexcm membrane. The cell-free broth is then concentrated by ultrafiltration with a 8,000 molecular weight cut-off membrane. The pH is adjusted to 5.5 with concentrated MES buffer (2-(N-morpholino)ethanesulfonic acid). The protease is further purified by cation exchange chromatography with S-sepharose and elution with NaCl gradients. See Scopes, R. K., xe2x80x9cProtein Purification Principles and Practicexe2x80x9d, Springer-Verlag, New York (1984).
A pNA assay (DelMar et al., Analytical Biochemistry, Vol. 99, pp. 316-320 (1979)) is used to determine the active protease concentration for fractions collected during gradient elution. This assay measures the rate at which p-nitroaniline is released as the protease hydrolyzes the soluble synthetic substrate, succinyl-alanine-alanine-proline-phenylalanine-p-nitroaniline (sAAPF-pNA). The rate of production of yellow color from the hydrolysis reaction is measured at 410 nm on a spectrophotometer and is proportional to the active protease moiety concentration. In addition, absorbance measurements at 280 nm are used to determine the total protein concentration. The active protease/total-protein ratio gives the protease purity, and is used to identify fractions to be pooled for the stock solution.
To avoid autolysis of the protease during storage, an equal weight of propylene glycol is added to the pooled fractions obtained from the chromatography column. Upon completion of the purification procedure the purity of the stock protease solution is checked with SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and the absolute enzyme concentration is determined via an active site titration method using trypsin inhibitor type II-T: turkey egg white (Sigma Chemical Company, St. Louis, Mo.).
In preparation for use, the protease stock solution is eluted through a Sephadex-G25 (Pharmacia, Piscataway, N.J.) size exclusion column to remove the propylene glycol and exchange the buffer. The MES buffer in the enzyme stock solution is exchanged for 0.01 M KH2PO4 solution, pH 5.5.
With the protease prepared it may be utilized for functionalization with one or more addition moieties to produce the protease conjugate. The precursor to the addition moiety (the precursor to the addition moiety reacts with the precursor to the protease moiety to form the protease conjugate which is comprised of the addition moiety and the protease moiety) is preferably activated to enhance reactivity with the precursor to the protease moiety. Such activation is well-known in the art. Non-limiting examples of methods of protease conjugate preparation are provided below.